1. Field of the Invention
The present invention relates to tumor targeting, and particularly to novel bleomycin conjugates for targeting a radioimaging or therapeutic compound to a body tumor.
2. References
The following references are referred to herein by corresponding number:
1. Umezawa, H., Pure Appl Chem (1970) 28: 665. PA0 2. Fujii, A., J., Antibiot (1973) 26: 398. PA0 3. Nouel, J. P., et al, Nouv Presse Med (1972) 2: 95. PA0 4. Goodwin, D. A., et al, Seminars in Nuc Med (1976) VI: 3. PA0 5. Goodwin, D. A. et al, In Radiopharmaceuticals II Proceedings of the Second International Conference on Rad, N. Y., Sodd, V. J., et al, eds, (1979) pp 275-284. PA0 6. Goodwin, D. A., et al, J Nuc Med (1981) 22: (9): 787. PA0 7. De Riemer, L. H., et al, J Med Chem (1979) 22: 1019. PA0 8. De Riemer, L. H., et al, J Lab Comps & Radpharm (1981) 18(10): 1517. PA0 9. Wensel, T. G., et al, in Radioimaging and Radioimmunotherapy, Burchiel, S. W., et al, eds, Elsevier, (1983) p 185 PA0 10. Chang, C.-H., et al, Biochem, Biophys Res Commun (1983) 110(3): 959. PA0 11. Kohler, B., et al, Nature (1975) 256: 495. PA0 12. Monoclonal Antibodies, Kennett, T. J., et al, eds Plenum (1980). PA0 13. Meares, C. F., et al, Proc Natl Acad Sci (USA) (1976) 73(11): 3803.
3. Background PA1 4. Summary of the Invention
Bleomycin is an anti-tumor antibiotic which has been shown to localize selectively within many types of tumors (References 1 and 2). Addition of .sup.57 Co to bleomycin yields a stable cobalt chelate whose use in radioimaging has been investigated (Reference 3). Although the chelate has been shown to provide good radioimaging capabilities, the relatively long half life of .sup.57 Co (270 days) creates unacceptable health and contamination problems. Other metal chelates of bleomycin, including .sup.111 In, .sup.64 Cu, .sup.67 Cu, .sup.99 Tc, .sup.62 Zn and .sup.67 Ga, that have been investigated lack the requisite in vivo stability for use in radioimaging (Reference 4).
The inventors have previously described a class of bleomycin-chelate compounds that can form stable metal chelates with a variety of metals, including .sup.111 In, that are clinically useful for radioimaging purposes. To prepare these compounds, an A.sub.2 form of bleomycin is isolated from a commercial preparation of bleomycin, which characteristically contains a mixture of bleomycin A.sub.2, B.sub.2, and minor amounts of demethylated A.sub.2 (DMA.sub.2). The isolated A.sub.2 form is demethylated to produce a thioether, which can react with a bifunctional chelating agent, such as p-bromoacetamidobenzyl-EDTA, to link the chelate to bleomycin through a sulfonium group. In vivo studies with model tumor systems in mice and with human cancer patients have demonstrated that the chelate compounds provide a useful radioimaging tool for determining size and location of many types of tumors (References 5-7).
The above-described bleomycin-chelate compounds, despite their demonstrated clinical usefulness in an experimental setting, have a number of practical limitations. The required bleomycin purification and demethylation steps mentioned above reduce product yield significantly and add to the overall preparation effort. The demethylation reaction may also lead to undesired side reactions (Reference 8). In terms of compound stability in vivo, it appears likely that the sulfonium group in the compound can be cleaved intracellularly, concurrently transferring the metal-chelate group to biomolecules in vivo, as evidenced by increased background levels in radioimaging applications.
It is therefore an important object of the present invention to provide novel bleomycin conjugates which overcome above-discussed limitations of prior art bleomycin compounds.
A more specific object of the invention is to provide such conjugates which can be prepared readily and at high yields from commercially available bleomycin preparations.
Another object is to provide such conjugates which are stable in vivo, and have uptake and biodistribution characteristics which are substantially the same as those of cobalt bleomycin.
Providing a method of making such conjugates, and for using the same in tumor diagnosis and therapy are other important objects of the invention.
The invention includes a bleomycin conjugate for use in targeting a compound to a body tumor. The conjugate comprises an oxidized bleomycin/cobalt(III) complex and the compound to be targeted, joined to the complex through a monodentate cobalt-sulfur coordinate bond. The bleomycin/cobalt(III) complex may include one or more bleomycin forms, including A.sub.2, DMA.sub.2 and B.sub.2 forms. In one general embodiment, the target compound is a chelating agent, with or without chelated metal, where the metal may be one which is useful for radioimaging or for therapeutic uses, either by itself or as a radiosensitizer (to be used with externally administered high-energy radiation). In another general embodiment, the compound is an anti-tumor drug or other therapeutic agent. The therapeutic compound may be joined to the bleomycin complex through a spacer arm having a labile bond, such as a disulfide bond, which can be cleaved intracellularly to release the drug from bleomycin after uptake into a tumor cell.
In a preferred method for forming the conjugate, an oxidized bleomycin-cobalt(III)-aquo complex is coupled, through a monodentate cobalt-sulfur coordinate bond, to a bifunctional thiol coupling agent. The target compound is attached to the complex through the free reactive end group of the coupling agent. The coupling agent thus forms a spacer arm between its coordinate-bond attachment to the bleomycin complex and the target compound.
The conjugate has tumor uptake and biodistribution characteristics which are very similar to those of cobalt bleomycin. Tumor-specific uptake can be enhanced severalfold by administering an antibody against the target compound prior to or contemporaneously with administering the conjugate to a patient. The enhanced tumor uptake appears to be due to the ability of antibody to sequester the relatively small conjugate in the bloodstream, and thereby prevent rapid renal clearance.
Further included in the invention are methods for using the novel conjugates for tumor radioimaging, therapeutic and radiosensitizing purposes.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying figures.